CN104645351A - Application of hsa-miR-487a in preparation of medicine for treating or preventing liver cancer after operation - Google Patents
Application of hsa-miR-487a in preparation of medicine for treating or preventing liver cancer after operation Download PDFInfo
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- CN104645351A CN104645351A CN201510038046.7A CN201510038046A CN104645351A CN 104645351 A CN104645351 A CN 104645351A CN 201510038046 A CN201510038046 A CN 201510038046A CN 104645351 A CN104645351 A CN 104645351A
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Abstract
The invention provides application of hsa-miR-487a in preparation of a medicine for treating or preventing liver cancer after an operation. The invention proves that the miRNA named hsa-miR-487a has high expression in the liver cancer for the first time through research, and by utilizing the characteristic of the RNA, the application of later-stage individual treatment is facilitated by adopting the hsa-miR-487a probe to predict the postoperative prognosis of a patient.
Description
Technical field
The present invention relates to the little miRNAs of a kind of endogenic non-coding, comprise its pharmaceutical composition, and it is used for the treatment of or the postoperative novelty teabag prevented liver cancer, especially, relate to a kind of hsa-miR-487a and be used for the treatment of or the postoperative purposes prevented liver cancer in medicine.
Background technology
As everyone knows, hepatocarcinoma (hepatocellular carcinoma, HCC, be called for short hepatocarcinoma) is the fifth-largest kinds of tumor of global male, has that prognosis is severe, mortality rate high, and the life of serious threat our people is with healthy.Be 7,483 ten thousand people as the world in 2008 increases hepatocarcinoma case newly, and the same year PLC mortality case up to 6,959 ten thousand people, its incidence and mortality is close to 1:1.Although in recent years along with the continuous progress of diagnostic imaging and surgical technic and take Sorafenib as the emerging of hepatoma-targeting medicine of representative, for the early diagnosis of hepatocarcinoma and treatment provide may with more and more effective therapeutic choice, make the treatment level of hepatocarcinoma obtain certain raising.
But take a broad view of current hepatocarcinoma clinical treatment present situation, the recurrence of PHC rate of transform still can be in any more, within postoperative 5 years, survival rate is still hovered about 30%, and the serious raising constraining hepatocarcinoma overall therapeutic level cannot make it the improvement obtaining essence.Trace it to its cause be exactly understand that hepatocarcinoma occurs not yet completely, development and the precise mechanism of Invasion and Metastasis.This seriously constrains the understanding of hepatocarcinoma progression and the research and development to new Hepatoma therapy medicine.Therefore, how to further investigate inquire into that HCC occurs, development and the mechanism of Invasion and Metastasis become in HCC research field now one very important scientific issues.
Microrna (microRNAs is called for short miRNAs) is that a class length is about 20 ~ 24nt, has the strand small RNA molecular of the endogenous non-coding protein of remarkable biological significance.MiRNA plays corresponding regulating and controlling effect in genetic transcription after-stage.Estimate according to existing document, miRNA can in body about 1/3 albumen synthesis play regulating and controlling effect, miRNAs can the adjustment of the different physiology of wide participation and pathological process, as growth, apoptosis, differentiation, propagation, metabolism, stress wait.
In view of miRNA is usually positioned the brittle point that chromosome is easy to loss, rearrangement, amplification, the chromosome brittle point part causing miRNA to locate is easy to occur disappearance, resets, increases, and then it is abnormal to cause miRNA to express appearance, and directly cause the functions such as cell proliferation, differentiation, transfer, apoptosis abnormal change also to occur thereupon, the appearance of inducing tumor cell.The function that miRNA is powerful and special generation, control methods have attracted the concern of numerous scholar, at present large quantity research has proved that miRNA all plays an important role in the generation of tumor, propagation, differentiation, invasion and attack, transfer, apoptosis, starts to show up prominently in the field such as diagnosis, prognosis evaluation, treatment of tumor.
Summary of the invention
For above-mentioned problems of the prior art, the present inventor has been found and hepatocarcinoma Invasion and Metastasis and the closely-related molecular mechanism of hepatocarcinoma breeding by research, and utilizing the microRNA played a crucial role in this molecular mechanism---the change of the expression of hsa-miR-487a and effect path, provide the pharmaceutical composition being used for the treatment of hepatocarcinoma.
According to an aspect of the present invention, provide a kind of pharmaceutical composition, it comprises micromolecule nucleic acid hsa-miR-487a.
Further, micromolecule nucleic acid hsa-miR-487a be connected with delivery vector after in pharmaceutical composition.
A progressive ground, delivery vector is morpholino nucleic acid.
According to a further aspect in the invention, the application of a kind of micromolecule nucleic acid hsa-miR-487a in preparation treatment or the postoperative medicine prevented liver cancer is additionally provided.
A progressive ground, application comprise micromolecule nucleic acid hsa-miR-487a is connected with delivery vector after be used in medicine.
A progressive ground, delivery vector is morpholino nucleic acid.
A progressive ground, medicine is trophophase for hepatocarcinoma or the medicine of hepatoma Metastasis phase.
A progressive ground, the diagnosing cancer of liver test kit containing micromolecule nucleic acid hsa-miR-487a.
According to a further aspect in the invention, additionally provide a kind of test kit for detecting hepatocarcinoma, test kit comprises micromolecule nucleic acid hsa-miR-487a.
The present invention has following beneficial effect:
1. the present invention confirms the effect path of hsa-miR-487a first by research, and has the characteristic that can promote the Invasion and Metastasis of hepatoma carcinoma cell and the effect of propagation.Utilize this characteristic, combined by the miRNA with this structure with existing pharmaceutical carrier, after administration, this medicine effectively can suppress the expression of this gene in animal body, thus suppresses propagation and the transfer of hepatoma carcinoma cell, thus reaches the object improving liver cancer patient prognosis.
2. the application of hsa-miR-487a provided by the invention includes treatment for hepatocarcinoma or postoperative prevention.
3. by research, the present invention confirms that the miRNA of hsa-miR-487a by name exists high expressed in hepatocarcinoma first, utilizes this characteristic of this RNA, may be used for preparing the test kit detecting potential liver cancer patient.
Except object described above, feature and advantage, the present invention also has other object, feature and advantage.Below with reference to figure, the present invention is further detailed explanation.
Accompanying drawing explanation
The accompanying drawing forming a application's part is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 illustrates hepatocarcinoma chip results schematic diagram;
Fig. 2 is shown into the schematic flow sheet including eliminating and grouping process in selecting 550 routine patients;
Fig. 3 shows the expression of hsa-miR-487a in hepatocarcinoma, wherein scheme A and show the expression of training queue He verifying hepatocarcinoma hsa-miR-487a in queue respectively with figure B, red line shows that in cancerous tissue, hsa-miR-487a is higher than cancer beside organism's 2 times of levels, to have in 85/132 and 43/66 routine sample cancerous tissue hsa-miR-487a expression respectively higher than more than cancer beside organism's twice in two queues; Figure C is shown in two queues, and in statistics display cancerous tissue, hsa-miR-487a expression is all significantly higher than cancer beside organism; Figure D is shown in three kinds of hypotypes of two queue hepatocarcinoma, and hsa-miR-487a expresses SHCC and the SLHCC group being significantly higher than low metastatic potential in the hypotype NHCC of high metastatic potential;
Fig. 4 shows hsa-miR-487a expression in liver cancer tissue and cancer beside organism.Hybridization in situ technique is adopted to detect hsa-miR-487a expression in the fixing section of liver cancer tissue paraffin.Figure A shows negative control in situ hybridization probe Scramble result figure, and figure B is shown as the in situ hybridization probe U6 result figure into internal reference and positive control, and figure C shows that employing hsa-miR-487a probe in detecting detects hsa-miR-487a expression in sample.In picture, blueness is in situ hybridization probe positive particle, and redness is the non-specific core dyestuff of core fast red.In figure, visible hsa-miR-487a is significantly higher than cancer beside organism at Expression In Hepatocellular Carcinoma, can see significant demarcation line between the two;
Fig. 5 shows hsa-miR-487a expression in normal liver cell system and hepatoma cell line.Realtime-PCR is adopted to detect primary hepatocyte system (PHH), normal liver cell system (L02) and hepatoma cell line (HepG2, PLC, Bel7402, MHCC97-L, SMMC7721, Huh7, Hep3B, MHCC97-H, HCCLM3) expression of hsa-miR-487a in cell line.Hsa-miR-487a expresses all higher than normal liver cell system in hepatoma cell line;
Fig. 6 shows that the patient of high expressed hsa-miR-487a in liver cancer tissue often has overall survival and the disease free survival rate of relative mistake.Figure A and figure B illustrate to train queue and overall survival and disease free survival rate in hsa-miR-487a height expression group in checking queue respectively.In hsa-miR-487a low expression group, patient has good postoperative overall survival and disease free survival rate;
Fig. 7 shows employing slow virus infection hepatoma cell line efficiency of infection.The bright visual field (Brightfield) shows finding under light microscopic, and green fluorescence (GFP) shows the cell of the infected slow virus of success under the same visual field.Stacking chart (Merge) is then the picture that obtains after being superposed with green fluorescence figure in the bright visual field, clearly can to see under appearance that feeling of success is caught an illness the cell of poison and uninfecting virus;
Fig. 8 shows the expression adopting wherein hsa-miR-487a after slow virus infection hepatoma cell line.Figure A shows that blank HCCLM3 cell line (untreated) and HCCLM3 infection of cell line hsa-miR-487a to intervene after viral (Anti-hsa-miR-487a) or negative control virus (Negative control, NC) wherein hsa-miR-487a expression.Figure B to show after blank HepG2 cell line (untreated) and HepG2 infection of cell line hsa-miR-487a process LAN virus (hsa-miR-487a) or negative control virus (Negative control, NC) wherein hsa-miR-487a expression;
Fig. 9 shows that hsa-miR-487a has the effect of the propagation promoting hepatoma carcinoma cell.Figure A shows 7 days cell proliferation curves, after visible intervention hsa-miR-487a expresses, (Anti-hsa-miR-487a) can suppress the multiplication capacity of hepatoma cell line HCCLM3, and can promote the multiplication capacity of hepatoma cell line HepG2 after process LAN hsa-miR-487a.Figure B left diagram Clone forming Test result, right diagram colony counts statistics, after intervening hsa-miR-487a expression, Cell clonality declines, Cell clonality enhancing after process LAN hsa-miR-487a expresses.Figure C left diagram cell cycle result figure, right graphical results cartogram, statistical result shows that hsa-miR-487a can promote that cell enters the G2/M phase by the G0/G1 phase;
Figure 10 shows the propagation adopting Edu dye marker proliferative cell to confirm hsa-miR-487a promotion hepatoma carcinoma cell.Figure AHoechest33342 is nucleus dyestuff, and labelling all cells nucleus is blue.Edu is thymidine analog, and the cell that labelling is in proliferating cycle is red.Overlay shows that, by Hoechest33342 and Edu figure superposition, red cell is for being in proliferation period cell, blue for being in the cell of quiescent stage.Figure B is the ratio that the cell being in proliferation period in figure A in each group accounts for total cellular score;
Figure 11 shows that hsa-miR-487a has and promotes the transfer of hepatoma carcinoma cell and the effect of transfer ability.Figure A shows that hsa-miR-487a promotes the transfer ability of hepatoma carcinoma cell.Left diagram Transwell result, right pictural statistics result.Figure B shows that hsa-miR-487a promotes the transfer ability of hepatoma carcinoma cell.Left diagram cut healing result, right pictural statistics result;
Figure 12 shows in body and suppresses hsa-miR-487a can reduce the growth of Tumor suppression in body and pulmonary shifts.Show each hepatoma cell line is expelled to different nude mice by subcutaneous on the left of figure A, put to death after one month and cut equal large tubercle block and be planted in another nude mice liver, then after January, put to death this photo of sampling.Right side shows each group of tumor size statistics.Visible suppression hsa-miR-487a can reduce the speed of growth of hepatocarcinoma tumor block, and process LAN hsa-miR-487a then accelerates its speed of growth.Figure B carries out hematoxylin-eosin staining method dyeing representative graph after showing the fixing section of nude mice lung tissue paraffin.In figure there is more Nodules relative in high expressed group lung tissue in visible hsa-miR-487a.Figure C shows the nude mice ratio occurring Intrahepatic metastasis in each group.Figure D shows in each group the nude mice ratio occurring transfer in lung;
Figure 13 shows that in tissue, hsa-miR-487a and ki67 expresses relevant.Adopt the expression of ability of cell proliferation mark Ki67 in Immunohistochemical detection nude mouse tumor.Figure A, B show the expression of ki67 in nude mice HCCLM3 group and HepG2 group liver cancer tissue.The figure high expressed hsa-miR-487a that lets others have a look at of B and lowly express ki67 expression in the liver cancer tissue of hsa-miR-487a.
Figure 14 shows Vivo-Morpholino schematic arrangement.It is Vivo-Morpholino structure that Morpholino molecule transfers to Gene Tool company to synthesize (the said firm official website is: http://www.gene-tools.com/) figure Middle molecule structural representation, and what wherein determine Morpholino function is the base sequence of B labelling in figure;
Figure 15 shows that Morpholino-Anti-hsa-miR-487a suppresses tumor proliferation and Lung metastases in liver.Adopt the HCC of Luciferase labelling to set up original position and become tumor model, after nude mice original position becomes tumor model to set up, Morpholino-Anti-hsa-miR-487a contrasts Morpholino-Control through tail vein injection twice weekly with it, carry out weekly living body fluorescent to detect once, put to death after 6 weeks and collect sample.Figure respectively organizes living body fluorescent testing result when A shows 6 weeks.In figure, in visible Morpholino-Control group, fluorescence is significantly better than Morpholino-Anti-hsa-miR-487a group.Figure B shows each group of living body fluorescent detection lump photon flow (Total Flux) weekly, and its value height represents the size of lump.Nude mice liver organization and tumor tissues in the left pictorial image A of figure C, figure C right diagram gross tumor volume cartogram.In figure, in visible Morpholino-Control group, Tumor size is obviously greater than Morpholino-Anti-hsa-miR-487a group.In the left pictorial image A of figure D, nude mice lung tissue living body fluorescent imaging results figure, right figure are the nude mice number cartogram occurring in each group of lung shifting.In figure in visible Morpholino-Control group Lung metastases number obviously more than Morpholino-Anti-hsa-miR-487a group.Figure E shows that each group of nude mice lung tissue paraffin is cut into slices after fixing, adopts hematoxylin-eosin staining method coloration result figure.
Detailed description of the invention
Below in conjunction with accompanying drawing, embodiments of the invention are described in detail, but the multitude of different ways that the present invention can be defined by the claims and cover is implemented.
Morpholino nucleic acid is Vivo-Morpholino delivery vector herein.Hsa-miR-487a is humanized's herein.
General introduction
Below in specific embodiment, first in embodiment 1 by include in from Xiang Ya hospital of Central South University 30 examples Xiang Ya hospital row hepatocarcinoma excision liver cancer tissue by the chip analysis three kinds of Exiqon company see cancerous tissue in and miRNAs express spectra in cancer beside organism.From this express spectra as shown in Figure 1, pick out the hsa-miR-487a being high expressed in 3 kinds of hepatocarcinoma hypotypes (SHCC, SLHCC, NHCC) and carry out follow-up research.In example 2, to the patient of the many cases row hepatectomy lump included in from Xiang Ya hospital of Central South University to fresh liver cancer tissue and corresponding contiguous non-tumor hepatic tissue, carry out prominent light real-time quantitative PCR to this hepatic tissue respectively to detect and hybridization in situ experiment, acquired results is listed in Fig. 3 ~ 5.Can find that income analysis result is identical with to the statistical result of sufferer liver cancer tissue in embodiment 1.All confirm that hsa-miR-487a is high expressed in various hepatoma carcinoma cell and patient tissue.
Embodiment 3 is by adding up the postoperative indices of liver cancer patient, and in organizing after finding operation in patients, the prognosis of hsa-miR-487a high expressors is poor, and then prognosis indices is better for the low expresser of hsa-miR-487a.Absolutely prove that hsa-miR-487a can reflect liver cancer patient prognosis rehabilitation efficacy, whether there is the possibility of cancer cell metastasis or regeneration.Acquired results is listed in Fig. 6 and form 1 ~ 7.Embodiment 4 couples of hsa-miR-487a carry out every experiment in vitro, to confirm its Proliferation Ability to hepatocarcinoma had and metastasis inhibition effect further.The results are shown in Fig. 7 ~ 11.Under the support of above experimental result, experiment in vivo is carried out to hsa-miR-487a.Experimentation and acquired results are as described in Example 5.By hsa-miR-487a is bonded on vivo-Morpholino in embodiment 5, in conjunction with after structure as shown in figure 14.N=23 in Figure 14 in hsa-miR-487a.Found by zoopery, in this way after administration, again can breed in liver or be transferred in its hetero-organization by effective anticancer, can be used for hepatocarcinoma early, in and late period every treatment in, especially can play the growth and transfer that suppress hepatoma carcinoma cell.Improve postoperative rehabilitation, prevent liver cancer recurrence.Simultaneously by embodiment 6, adopt conventional method by hsa-miR-487a generate a reagent box, can use it for and detect the high expressed whether postoperative patient or potential liver cancer patient exist this gene, to detect whether patient suffers from hepatocarcinoma.Also can be used for detecting postoperative patient whether liver cancer recurrence and/or hepatoma Metastasis.
An aspect of of the present present invention provides a kind of pharmaceutical composition, wherein containing micromolecule nucleic acid hsa-miR-487a.Known by the every experiment in above-described embodiment, micromolecule nucleic acid hsa-miR-487a has the effect suppressing hepatoma cell proliferation and transfer.After being combined with suitable carrier by hsa-miR-487a, can animal body be acted on, and then play the therapeutic effect to hepatocarcinoma.Obvious hsa-miR-487a also can add other to be had in the medicine of Hepatoma therapy effect, plays the effect increasing its curative effect or its performance curative effect collaborative.The administering mode of hsa-miR-487a plays can make it effect suppressing hepatoma cell proliferation and transfer.Hsa-miR-487a can combine with any conventional drug administration carrier of gene, thus the growth now inhibitory action to hepatoma carcinoma cell in patient body.
Micromolecule nucleic acid hsa-miR-487a has the sequence shown in SEQ ID NO:1.
Preferred delivery vector is morpholino nucleic acid.Administration verified effect animal body to suppression transfer and propagation in embodiment 5 is carried out with this carrier.
Another aspect of the present invention additionally provides the application of a kind of micromolecule nucleic acid hsa-miR-487a in the medicine preparing Hepatoma therapy.Can there is high expressed in known hsa-miR-487a in the above-described embodiments in liver cancer patient tissue or hepatoma carcinoma cell.This high expressed, may be used for preparing the test kit with detection effect.This test kit can, by detecting tissue, by this tissue of pcr amplification, to obtain the expression of wherein hsa-miR-487a, thus be determined to provide the human body of this given the test agent whether to there is hepatocarcinoma.Thus improve the examination of early hepatocarcinoma, being beneficial to sufferer can obtain medical treatment as early as possible, in order to avoid delay treatment.Certainly this application also comprises the medicine containing hsa-miR-487a group or its moieties, after administration, reaches the effect suppressing hepatoma cell proliferation or transfer, thus reaches the application of the effect of Hepatoma therapy.
Preferred micromolecule nucleic acid hsa-miR-487a has the sequence shown in SEQ ID NO:1.The nucleic acid of this sequence, Detection results is more accurate.
Preferred micromolecule nucleic acid hsa-miR-487a is made into the medicine be connected with delivery vector.By the conventional method that administered vehicle is nucleic acid administration, this administering mode can be viral vector, cholesterol, chitosan or liposome.Be preferably the drug administration carrier of nanoparticle level.Be more preferably morpholino nucleic acid.Now stable curative effect after administration.
The medicine of gained in the application of micromolecule nucleic acid hsa-miR-487a in medicine, may be used for treating following case: the trophophase of hepatocarcinoma or hepatoma Metastasis phase.The liver cancer growth phase herein comprises early stage, mid-term and the late period of onset of liver cancer.The hepatoma Metastasis phase refers to the transfer comprising any interim generation of hepatocarcinoma in trophophase in the period that hepatocarcinoma generation is shifted, this phase.
Hsa-miR-487a also can be used in the prevention of prevention of postoperative liver cancer recurrence and transfer.By human administration, can the effectively expression of hsa-miR-487a in inhibition test animal body, thus reach the effect suppressing liver cancer recurrence and transfer.Can see embodiment 5.
Embodiment
In following embodiment, the formula of agents useful for same can referring to miRCURY-LNA-microRNA-ISH-Optimization-Kit-manual.pdf 14-15 page Table2 and Table3.
In following embodiment, patient includes in and carries out all like this.Be included into from the patient of 550 customary hepatoncus excisions of Xiang Ya hospital of Central South University in January, 2003 to 2013 year December, wherein after 32 routine operation in patients, sick inspection turns out to be cancer of biliary duct eliminating, 54 examples turn out to be hemangioma and are excluded, remain 444 routine patients with hepatocellular carcinomas and before in January, 2008 with behind 2008 1 and the moon, be divided into two groups of data according to its operating time, the routine patient of expection employing 200 studies, training queue (Training cohort) and checking queue (Validation cohort) is entered according to 2:1 ratio sample drawn. wherein train the final female of queue to enter patient 132 example, patient 66 example is included in checking queue in.
Edu cell proliferation test is undertaken by adopting the concrete operation step in Edu test kit in appended description.
Vivo-Morpholino synthesis and Vivo-Morpholino Design and synthesis process can with reference to contents disclosed in the pertinent literature of list of references part.
The hsa-miR-487a adopted in the present invention the Morpholino sequence suppressing it to act on are: Morpholino-Anti-hsa-miR-487a:GATGTCCCTGTATGATTCGTCAT.
The independent sequence of hsa-miR-487a is as shown in SEQ ID NO.1.Hsa-miR-487a Mismatch controls sequence is: Morpholino-Control:GtTcTCCgTGTATcATTCcTCAT as shown in SEQ ID NO.2 (illustrated with lower case by base mismatch for ease of distinguishing, real is capitalization).Can be found by contrast SEQ ID NO.1 and SEQ ID NO.2, after only changing several base sequence, gained miR does not just have various effect provided by the invention and performance.
Below in research with hsa-miR-487a mispairing RNA (SEQ ID NO.2) for training queue, the effect that hsa-miR-487a (SEQ ID NO.1) has to be described by approximate base sequence.Morpholino-Anti-hsa-miR-487a molecular weight is 9580OD:467.82.
Embodiment 1
CDNA microarray
First adopt the miRCURYTM Array microarray kit V8.0 chip of Exiqon company to analyze the liver cancer tissue of 30 examples at Xiang Ya hospital row hepatocarcinoma excision, detect the miRNAs express spectra in the cancerous tissue of solitary large HCC (SLHCC), small liver cancer (SHCC) and nodositas hepatocarcinoma (NHCC) and cancer beside organism respectively.By contrasting the miRNA express spectra of different hepatocarcinoma hypotype, find that the miRNAs taking up differential expression in different subtype hepatocarcinoma and cancer side in cancer and cancer in a large number composes.Wherein comparatively cancer beside organism is high at Expression In Hepatocellular Carcinoma for hsa-miR-487a, and its expression high expressed in high Invasion and Metastasis hypotype NHCC, and SLHCC and the SHCC that transfer ability is more weak, result is see Fig. 1.Adopt the screening of miRNA chip technology to find that hsa-miR-487a is high expressed in 3 kinds of hepatocarcinoma hypotypes (SHCC, SLHCC, NHCC), express obviously high compared with other two groups in the hepatocarcinoma hypotype NHCC that transitivity is stronger.SHCC, small liver cancer (tumor size <5cm).SLHCC, solitary large HCC (tumor size >=5cm, single tumor nodule) NHCC, nodositas hepatocarcinoma (tumor nodule number >=2).
Embodiment 2
Fresh liver cancer tissue and corresponding contiguous non-tumor hepatic tissue (Adjacent nontuinorous liver tissue, ANLT, apart from mass edge 1.0cm) the prominent light real-time quantitative PCR for miRNA expression is detected.40 routine small liver cancers are comprised, 40 routine nodositas hepatocarcinoma and 40 routine solitary large HCCs in this 120 routine fresh HCC specimen.The characteristic that the solitary large HCC defined has shows as single lump, peplos is had to be formed, volume comparatively large (diameter of tumor >5.0cm) but relatively less generation vein are invaded, after excision, recurrence is lower with the rate of transform, surgical effect and prognosis final result are all better, have unique clinical and molecular pathology feature and relative tumor biological behavior preferably.Diameter of tumor S5.0cm is then small liver cancer, and lump tuberosity number ^2 person is nodositas hepatocarcinoma [4].Pay special attention to when making a collection of specimens to avoid hemorrhagic necrosis tissue, namely specimen cuts relevant portion tissue specimen and is sub-packed in sterilizing EP pipe in vitro and be then placed in liquid nitrogen rapidly, is stored in-80 DEG C of ultra cold storage freezers for subsequent use subsequently.Ultra cold storage freezer model is Forma 702, purchased from American Thermo Scientific.
Hybridization in situ experiment method and instrument thereof: the hsa-miR-487a of two digoxigenin labeled, scramble, U6 probe is bought from Exiqon company of Denmark, the miRCURY LNA that the test kit used is Exiqon company
tMmicroRNA ISH Optimization Kit (FFPE) #90000.The fixing section of paraffin adopt dimethylbenzene, 99.9%, 96%, 70% ethanol repeatedly soaks dewaxing.The E.C. 3.4.21.64 37 DEG C adding 15 μ g/mL hatches 10 minutes, again adopts 70%, 96% after cleaning, and the ethanol gradient of 99.9% soaks dehydration.Add the probe through 90 DEG C of 4 minutes degenerative treatments, U6 concentration and probe concentration 1nM, other concentration and probe concentration 20nM, 50-60 DEG C hatch 1 hour, at the 5x of 50 DEG C, soak step by step in 1x, 0.2xSSC buffer, each 5 minutes.15 minutes are hatched with the confining liquid (Antibody blocking solution) containing sheep blood serum.Adopt anti digoxin antibody (Sheep-anti-DIG-AP) 1:800 concentration room temperature to hatch 60 minutes, add AP substrate 30 DEG C after phosphate Tween buffer (PBS-T) cleaning and hatch 2 hours.Employing is hatched stop buffer KTBT and is hatched cessation reaction, and uses water to clean 2 each 1min.Afterwards, adopt core fast red dyes nucleus 1 minute, rinse 10 minutes at tap water down-flow water.The dehydration mentioned before repetition, adopts mounting medium to fix, avoids air-dry in omnidistance hybridization step.Microscopic examination can be adopted to take pictures after fixing.
Hsa-miR-487a is high expressed analysis in HCC tissue
In order to verify the expression of hsa-miR-487a in hepatocarcinoma further, be divided into training queue and checking queue two separate queues according to randomly drawing 444 described in test method to its operating time of sample evidence carrying out the patient of hepatocarcinoma excision for 2003 to 2013 in Xiangya Hospital, Central-South China Univ., process as shown in Figure 2.Training queue (Training cohort) and checking queue (Validtion cohort) two separate queues are adopted to confirm the expression of hsa-miR-487a in Tissues of Hepatocellular Carcinoma and prognosis significance in research.
Employing realtime-PCR technology for detection respectively organizes the expression in liver cancer tissue and cancer dish tissue, hsa-miR-487a expression adopts log2 (cancerous tissue expression/cancer beside organism's expression) to represent, in Fig. 3 A visible hsa-miR-487a training queue in 85/132 (65.39%), checking queue in 43/66 (65.15%) Expression In Hepatocellular Carcinoma higher than more than cancer beside organism's twice.In statistics display two queues, in liver cancer tissue, hsa-miR-487a expression is all significantly higher than cancer beside organism (P<0.01).In order to verify microRNA chip results, by small liver cancer in individual queue (SHCC), solitary large HCC (SLHCC), in nodositas hepatocarcinoma (NHCC), hsa-miR-487a expression is added up, the statistics display shown in Fig. 3 D is consistent with chip results, the expression of hsa-miR-487a in nodositas hepatocarcinoma is significantly higher than small liver cancer and solitary large HCC group, and the expression of hsa-miR-487a in small liver cancer and solitary large HCC not significant difference.In order to the expression of hsa-miR-487a in liver cancer tissue more can be got information about, have employed hybridization in situ technique to fix section to liver cancer tissue paraffin and detect, in Fig. 4, in the visible liver cancer tissue of result, hsa-miR-487a expresses and is significantly higher than cancer beside organism, has obvious differential expression between liver cancer tissue and cancer beside organism.Equally, also have chosen 9 strain hepatoma cell line (HepG2, PLC, Bel7402, MHCC97-L, SMMC7721, Huh7, Hep3B, MHCC97-H, HCCLM3) and 2 strain normal liver cell system (PHH, L02) relatime-PCR is adopted to detect the expression of wherein hsa-miR-487a, result as shown in Figure 5, same finds that in hepatoma carcinoma cell, hsa-miR-487a expression is all far above normal liver cell system level, with find in patient tissue consistent.
Embodiment 3
High expressed hsa-miR-487a is relevant with the poor prognosis of hepatocarcinoma
In order to whether postoperative with liver cancer patient the prognosis understanding hsa-miR-487a be relevant, whether be divided into high expressed group and low expression group higher than cancer beside organism's twice according to hsa-miR-487a expression in individual queue, the liver cancer patient Follow-up After data that oneself builds in conjunction with the present invention are analyzed two groups of patient's pathologic conditions and prognosis situation.First, compare the basic clinical data of two queues, to guarantee that the patient included in two queues has comparability, comparison result is listed in table 1.
Table 1 trains queue to compare with each clinical case feature in checking queue
In order to understand the relation between hsa-miR-487a expression and each clinical case feature, carry out correlation analysis, analysis result shows expression and tumor size, the tumor nodule number of hsa-miR-487a in two queues, with or without peplos, Microvascular invasion, TNM is by stages relevant, and in training queue, hsa-miR-487a and clinical case feature correlation are listed in table 2.In checking queue, the situation of hsa-miR-487a and clinical case feature correlation is listed in table 3.
Table 2. trains hsa-miR-487a and clinical case feature correlation in queue
Table 3. verifies hsa-miR-487a and clinical case feature correlation in queue
And these clinical case features known with Liver Cancer Operation after transfer closely related with recurrence.Therefore, it can thus be appreciated that hsa-miR-487a expression may be relevant with the transfer and relapse after Liver Cancer Operation.
In order to the relative risk (RR) that the expression exploring each clinical pathologic characteristic and hsa-miR-487a affects overall survival, disease free survival rate after Liver Cancer Operation, understand the independent hazard factor whether hsa-miR-487a is hepatocarcinoma post-operative survival rates or prognosis.COX regression model is adopted to carry out single multifactor statistical analysis to each variable in two queues.Statistical result showed: in training queue, liver cirrhosis, tumor nodule number, Microvascular invasion and hsa-miR-487a high expressed are the independent hazard factor of overall survival after Liver Cancer Operation, and liver cirrhosis, tumor nodule number, Microvascular invasion and hsa-miR-487a high expressed are equally also found to be the independent hazard factor of disease free survival after Liver Cancer Operation.Aforementioned result is listed in table 4 and table 5 respectively.
Table 4. single factor test and multiplicity train the risk factor of hepatocarcinoma overall survival in queue
Table 5 single factor test and multiplicity train the risk factor of hepatocarcinoma disease free survival rate in queue
In order to confirm that this research finds, in the checking queue with longer follow up time, again carried out analytic statistics equally, result be presented in checking queue, independent hazard factor that tumor nodule number, Microvascular invasion and hsa-miR-487a high expressed are overall survival after Liver Cancer Operation.See table 6.And liver cirrhosis, Microvascular invasion and hsa-miR-487a high expressed are the independent hazard factor of disease free survival after Liver Cancer Operation.See table 7.
Table 6. single factor test and multiplicity verify the risk factor of hepatocarcinoma overall survival in queue
Table 7 single factor test and multiplicity verify the risk factor of hepatocarcinoma disease free survival rate in queue
From table 6 and 7, listed result can be learnt, the high expressed of hsa-miR-487a usually and the overall survival of hepatocarcinoma and disease free survival rate closely bound up, hsa-miR-487a high expressed is one of important independent hazard factor of the low overall survival of hepatocarcinoma and low disease free survival rate.
In order to observe hsa-miR-487a high expressed and the low long-term postoperative prognosis expressing patient of hsa-miR-487a more intuitively, Kaplan-meier curve is adopted to carry out com-parison and analysis to the patient of hsa-miR-487a high expressed group and low expression group in two queues, statistic curve such as Fig. 6 A shows, and in training queue, hsa-miR-487a low expression group overall survival rate and disease free survival rate are all significantly better than hsa-miR-487a high expressed group.Equally, as Fig. 6 B shows, also in the checking queue with longer follow up time, this discovery is confirmed.
In liver cancer tissue, the patient of high expressed hsa-miR-487a often has a relatively poor long-dated survival prognosis thus.
Embodiment 4
Respectively following experiment in vitro is carried out to hsa-miR-487a, to confirm its effect to hepatocarcinoma further.
1. build cell line and be used for cell assay in vitro
After clear and definite high expressed hsa-miR-487a this situation relevant to the poor prognosis of hepatocarcinoma, it is desirable to understand further the reason that hsa-miR-487a causes patient's poor prognosis.As shown in Figure 5, the hepatoma cell line HepG2 that hsa-miR-487a expresses the highest hepatoma cell line HCCLM3 minimum with expression carries out follow-up study.Adopt slow virus carrier to divide hsa-miR-487a in cell to intervene and process LAN, set up the hepatoma cell line HepG2hsa-miR-487a of stably express hsa-miR-487a and the hepatoma cell line HCCLM3Anti-hsa-miR-487a cell line of stabilization checking hsa-miR-487a.After cell infection slow virus, adopt the method determination efficiency of infection that light microscopic and green fluorescence (GFP) combine, as shown in Figure 7, visible HCCLM3 and HepG2 respectively organizes cell infection efficiency and reaches more than 90%.Simultaneously, realtime-PCR is adopted to detect the expression of hsa-miR-487a in the stable cell line built, result as shown in Figure 8, visible in figure, hsa-miR-487a expression in Anti-hsa-miR-487a slow virus effective prevention hepatoma cell line HCCLM3, hsa-miR-487a slow virus effectively raises hsa-miR-487a expression in hepatoma cell line HepG2.In HCCLM3Anti-hsa-miR-487a, the expression efficiency of hsa-miR-487a is obviously suppressed, and in HepG2hsa-miR-487a cell line, hsa-miR-487a expresses and obviously increases.
2.hsa-miR-487a can promote the in-vitro multiplication ability of hepatoma carcinoma cell
Whether the multiplication capacity of hepatoma carcinoma cell often has close contacting with the growth of hepatocarcinoma, relevant with the multiplication capacity of hepatocarcinoma in order to understand hsa-miR-487a, have employed multiple test and confirms this situation.Cell growth curve such as Fig. 9 A shows, and after pounding out hsa-miR-487a, the multiplication capacity of hepatoma carcinoma cell HCCLM3 significantly reduces, and after process LAN hsa-miR-487a, the multiplication capacity of hepatocellular carcinoma H22 significantly improves.As Fig. 9 B shows, after hsa-miR-487a is pounded out in cell clone test display, the Clone formation digital display work of hepatoma carcinoma cell HCCLM3 reduces, and after process LAN hsa-miR-487a, clone's number of hepatocellular carcinoma H22 significantly improves.These two tests all confirm that the multiplication capacity of hsa-miR-487a and hepatocarcinoma has obvious relation.In order to understand hsa-miR-487a actually by affect in cell cycle which in stage thus affect the multiplication capacity of hepatoma carcinoma cell, flow cytometer cell cycle is adopted to detect, as shown in Fig. 9 C, after hsa-miR-487a is intervened in result display, hepatoma carcinoma cell G1 phase cell number increases, after process LAN hsa-miR-487a, its cell number of hepatoma carcinoma cell G1 reduces.Result shows, and hsa-miR-487a can promote that hepatoma carcinoma cell enters S phase or G2/M phase thus the final multiplication capacity promoting cell from the G1 phase.Simultaneously, thymidine analog Edu is also adopted to carry out the cell that labelling is in proliferating cycle, and adopt non-specific cell core dyestuff Hoechst33342 to carry out all nucleus of labelling, in the hope of observing the ratio being in the cells on total cells number of proliferation period more intuitively, result is shown in Figure 10, in figure, the visible hsa-miR-487a of intervention is in the cell number minimizing of proliferation period after expressing, and process LAN hsa-miR-487a is in proliferation period cell number after expressing increases.Display is in proliferation period HCCLM3 cell number after intervening hsa-miR-487a significantly reduces, and the HepG2 cell number being in proliferation period after process LAN hsa-miR-487a significantly increases.
Above result all confirms, hsa-miR-487a can play the effect promoting hepatoma carcinoma cell in-vitro multiplication ability.
3.hsa-miR-487a promotes vitro invasion and the transfer ability of hepatoma carcinoma cell
The invasive ability of hepatocarcinoma and the postoperative recurrence of hepatocarcinoma are closely related with transfer, in order to whether clear and definite hsa-miR-487a is relevant to the relapse and metastasis ability of hepatocarcinoma, have employed Transwell test and cut healing assay detects.The display of Transwell result of the test is with 11A, to intervene after hsa-miR-487a under Transwell film cell number comparatively matched group reduce and after process LAN hsa-miR-487a cell number comparatively matched group increase, prompting hsa-miR-487a can promote the invasive ability of hepatoma carcinoma cell.The display of cut healing result, after intervening hsa-miR-487a, 24h cell cut healing ability comparatively matched group obviously declines, process LAN hsa-miR-487a group 24h cell cut healing ability then comparatively matched group obviously promote, prompting hsa-miR-487a relevant with the ability of migrating of hepatoma carcinoma cell.These two equal susceptible of proofs of result of the test, miR-487 can promote vitro invasion and the transfer ability (Figure 11 B) of hepatoma carcinoma cell.After in Figure 11, hsa-miR-487a expression is intervened in result display, cell transfer declines with transfer ability, and process LAN hsa-miR-487a expresses rear transfer and strengthens with transfer ability.
Embodiment 5
Carry the Vivo-Morpholino animals administer method of miR-487: adopt tail vein injection administration, first nude mice being fixed on mice before injection fixes in syringe, afterbody is exposed to outside instrument, adopt 75% ethanol wiping nude mice afterbody repeatedly, play the effect of sterilization and blood vessel dilating, inject after adopting 1ml syringe to draw appropriate 15ug/ul Vivo-Morpholino after nude mice two sides of tail tail venous engorgement, administration total amount is pressed 12mg/kg and is calculated.
Animals administer and the mode of putting to death: Per-Hop behavior twice.Every day, whether routine observation nude mice survival condition, developed complications, and regularly adopts weekly living body fluorescent to observe tumor growth situation, put to death and get tissue after 6 weeks.
Living body fluorescent cell line builds: adopt Luciferase (LUC Photinus pyralis LUC Photinus pyralis FL) labelling HCCLM3 hepatoma carcinoma cell.Cell construction transfers to Shanghai Berthold company to build, and the LM3-GFP/mCherry-luc human liver cancer cell description that concrete building process can provide referring to the said firm is carried out.
Living body fluorescent imaging: Luciferase fluorogenic substrate is provided by Shanghai Berthold company.DPBS (Du formula PBS) is adopted to dilute fluorogenic substrate to 15mg/ml, degerming by 0.2 μm of strainer filtering.First adopt the pentobarbital sodium 100ul intraperitoneal injection of anesthesia nude mice of 2% before test, adopt 1ml syringe to draw fluorogenic substrate and carry out lumbar injection, administration total amount is 150mg/kg.Hunan University's chemical-biological sensing and meterological National Key Laboratory IVIS lumina II detection system is utilized to detect after 7-8 minute.Detect weekly once, postoperative recovery continues cultivation.After within 6th week, detecting, nude mice is anaesthetized execution deeply, taking-up lung tissue again adopts IVIS lumina II detection system to take pictures and observes pulmonary's metastasis rapidly.
1.hsa-miR-487a promotes tumor growth and the transfer ability of hepatoma carcinoma cell
In order to the liver cancer growth situation in human body better can be simulated, adopt the hepatoma cell line of above-mentioned stable transfection hsa-miR-487a intervention or process LAN to construct nude mice original position and become tumor model, often organize each 6 nude mices, after 1 month, by sacrifice, get liver and lung tissue is observed, after Figure 12 A shows 1 month, hsa-miR-487a intervention group hepatocarcinoma lump is significantly less than its matched group, its lump volume only has about 1/4 of matched group, and the obviously comparatively matched group increase of hsa-miR-487a process LAN hepatocarcinoma lump, its lump volume is matched group more than 5 times.Meanwhile, get its lung tissue after paraffin is fixed and cut into slices, adopt HE dyeing Microscopic observation, schematic diagram 12B shows in HCCLM3 matched group and has occurred a large amount of Nodules in nude mice lung tissue, and after intervening hsa-miR-487a, in lung, rarely metastasis occurs.Through statistics display, hsa-miR-487a intervention group Intrahepatic metastasis and metastasis in lung all comparatively contrast number and reduce, and Figure 12 C, D show the equal showed increased of metastasis in hsa-miR-487a process LAN group Intrahepatic metastasis and lung.The display of above result, hsa-miR-487a can promote hepatoma carcinoma cell growth in vivo and transfer ability.And hsa-miR-487a relative to all have in high expressed group more nude mice occurred in liver with Nodules in lung.
2. in liver cancer tissue hsa-miR-487a expression and its multiplication capacity closely related.
In order to confirm the relation of hsa-miR-487a expression and Hepatocarcinoma Proliferation ability in the tissue, first animal is adopted to test, the nude mice tumor tissue paraffin of the nude mice tumor tissue and the low hsa-miR-487a of expression that have chosen high expressed hsa-miR-487a carries out multiplication capacity mark Ki67 Immunohistochemical detection after fixing and cutting into slices, representative picture is as Figure 13 A, B shows Ki67 expression in the hepatocellular carcinoma in nude mice tissue of the more relative high expressed hsa-miR-487a of Ki67 expression in the hepatocellular carcinoma in nude mice tissue of relatively low expression hsa-miR-487a obviously to be reduced, in the liver cancer tissue of prompting high expressed hsa-miR-487a, comparatively hsa-miR-487a low expression liver cancer tissue is strong for ability of cell proliferation.Show ability of cell proliferation in the liver cancer tissue of high expressed hsa-miR-487a to increase compared with the expression of mark Ki67 simultaneously.
Equally, in human trial, according to the result of realtime-PCR as Fig. 3 A, B shows, people's hepatocarcinoma sample of the people's hepatocarcinoma sample and the low hsa-miR-487a of expression of choosing high expressed hsa-miR-487a with in situ hybridization (Fig. 4) result carries out the Immunohistochemical detection of multiplication capacity mark Ki67, result shows equally, and in the human liver cancer tissue of high expressed hsa-miR-487a, the expression of Ki67 is higher than the human liver cancer tissue of low expression hsa-miR-487a.Result is pointed out, and the lower expression group of liver cancer tissue propagation of high expressed hsa-miR-487a is strong.
3. intervene for targeting the Vivo-Morpholino that in body, hsa-miR-487a expresses to build
It is relevant with the postoperative poor prognosis of liver cancer patient that early-stage Study result has shown high expressed hsa-miR-487a, hsa-miR-487a all can promote propagation and the transfer ability of hepatoma carcinoma cell in vivo and in vitro significantly, and carry out intervening to it and can suppress the Proliferation and invasion transfer ability of hepatoma carcinoma cell significantly, the discovery prompting hsa-miR-487a of this effect may become a kind for the treatment of in hepatocarcinoma as a potential drug target or improve the effective medicine of height of patient's Postoperative determination.In view of Morpholino carrier medicament is at present for Ebola virus, Marburg virus, the treatment of Du Shi amyotrophy medicine, it is intervention techniques in body safely and effectively in a kind of body, Vivo-Morpholino is a kind of Morpholino carrier through modifying, the Morpholino medicine worked can effectively proceed in active somatic cell through intravascular injection or intraperitoneal injection by it, is a kind of Morpholino carrier having extremely strong utilization prospect.Therefore, entrusting Gene-tool company to construct, targeting intervenes hsa-miR-487a expresses in body Vivo-Morpholino (Morpholino-Anti-hsa-miR-487a) and its mispairing contrasts Vivo-Morpholino (Morpholino-Control), and Vivo-Morpholino structural representation and specific base sequence thereof are as shown in figure 14.In order to verify Morpholino-Anti-hsa-miR-487a in vivo to the therapeutic effect of hepatocarcinoma, again adopt the hepatoma carcinoma cell HCCLM3 of LUC Photinus pyralis LUC Photinus pyralis FL (Luciferase) labelling to construct 12 nude mice original positions and become tumor model, random assortment is two groups, Morpholino-Anti-hsa-miR-487a and Morpholino-Control injection is given respectively twice weekly through tail vein, medicine total amount calculates according to 12mg/kg, and administration concentration is 15ug/ul.Living body fluorescent detection system is regularly adopted weekly to detect nude mice tumor growth in vivo situation.Testing result is shown in Figure 15 A, in Figure 15 B, after display Morpholino-Anti-hsa-miR-487a administration, decreased tumor growth in nude mouse, Figure 15 C shows, after 6 weeks Morpholino-Anti-hsa-miR-487a injection group obviously comparatively matched group reduce, that particularly evident is Figure 15 D, to show in E in Morpholino-Anti-hsa-miR-487a injection group lung metastasis number and size all comparatively matched group significantly reduce.Result of study shows, and effectively can suppress the speed of growth of nude mice in-vivo tumour, reduce its pulmonary's metastasis after Morpholino-Anti-hsa-miR-487a application, is a kind of effective cancer treatment drug.
Occur in visible Morpholino-Control group in Figure 15 that more Pulmonary metastasis focuses Morpholino-Anti-hsa-miR-487a group does not then occur obvious Pulmonary metastasis focuses.
Embodiment 6
By method disclosed in CN200810143294.8 the wherein EG FL7 gene hsa-miR-487a replaced with in the present invention used can be obtained containing hsa-miR-487a be used for detect hepatocarcinoma detection kit.
Acquired results can detect the test kit of gained containing hsa-miR-487a by method disclosed in CN200810143294.8.Acquired results confirms that hsa-miR-487a can be used for the liver cancer patient detecting hsa-miR-487a high expressed.
Human liver cancer cell source can be LM3-GFP/mCherry-luc human liver cancer cell.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Above experimental technique and background technology part can with reference to Publication about Document related contents:
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Claims (9)
1. a pharmaceutical composition, is characterized in that, it comprises micromolecule nucleic acid hsa-miR-487a.
2. pharmaceutical composition according to claim 1, is characterized in that, in described pharmaceutical composition after described micromolecule nucleic acid hsa-miR-487a is connected with delivery vector.
3. pharmaceutical composition according to claim 2, is characterized in that, described delivery vector is morpholino nucleic acid.
4. the application of micromolecule nucleic acid hsa-miR-487a in preparation treatment or the postoperative medicine prevented liver cancer.
5. application according to claim 4, is characterized in that, described application comprise described micromolecule nucleic acid hsa-miR-487a is connected with delivery vector after be used in described medicine.
6. application according to claim 4, is characterized in that, described delivery vector is morpholino nucleic acid.
7. application according to claim 4, is characterized in that, described medicine is trophophase for hepatocarcinoma or the medicine of hepatoma Metastasis phase.
8. application according to claim 4, is characterized in that, the diagnosing cancer of liver test kit containing described micromolecule nucleic acid hsa-miR-487a.
9. for detecting a test kit for hepatocarcinoma, it is characterized in that, described test kit comprises micromolecule nucleic acid hsa-miR-487a.
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| CN1272709A (en) * | 1999-05-04 | 2000-11-08 | 许俊甫 | High-efficiency high-torsion high-support external rotor motor |
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